Modified Kappa Light Chain-Binding Polypeptides

ABSTRACT

The invention discloses kappa light chain-binding polypeptide comprising a mutated binding domain of  Peptostreptococcus  protein L, wherein at least one asparagine residue of a parental domain defined by, or having at least 95% or 98% sequence homology with, SEQ ID NO: 2-6 or 2 has been mutated to another amino acid residue which is not asparagine, proline or cysteine.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to the field of affinity chromatography,and more specifically to polypeptides comprising kappa lightchain-binding domains of Protein L, which are useful in affinitychromatography of many types of immunoglobulins and immunoglobulinfragments. The invention also relates to separation matrices containingthe polypeptides and to separation methods using such separationmatrices.

BACKGROUND OF THE INVENTION

Immunoglobulins and immunoglobulin fragments represent the mostprevalent biopharmaceutical products in either manufacture ordevelopment worldwide. The high commercial demand for and hence value ofthis particular therapeutic market has led to the emphasis being placedon pharmaceutical companies to maximize the productivity of theirrespective manufacturing processes whilst controlling the associatedcosts.

Affinity chromatography, typically on matrices comprising staphylococcalProtein A or variants thereof, is normally used as one of the key stepsin the purification of intact immunoglobulin molecules. The highlyselective binding of Protein A to the Fc chain of immunoglobulinsprovides for a generic step with very high clearance of impurities andcontaminants.

For antibody fragments, such as Fab, single-chain variable fragments(scFv), bi-specific T-cell engagers (BiTEs), domain antibodies etc.,which lack the Fc chain but have a subclass 1,3 or 4 kappa light chain,matrices comprising Protein L derived from Peptostreptococcus magnus (BÅkerström, L Björck: J. Biol. Chem. 264, 19740-19746, 1989; W Kastern etal: J. Biol. Chem. 267, 12820-12825, 1992; B H K Nilson et al: J. Biol.Chem. 267, 2234-2239, 1992 and U.S. Pat. No. 6,822,075) show greatpromise as a purification platform providing the high selectivityneeded. The Protein L disclosed in U.S. Pat. No. 6,822,075 comprises theamino acid sequence SEQ ID NO: 1 plus an additional AVEN sequence at theN-terminus.

(Protein L) SEQ ID NO: 1  KEETPETPETD SEEEVTIKAN LIFANGSTQT AEFKGTFEKATSEAYAYADT LKKDNGEYTV DVADKGYTLN IKFAGKEKTPEEPKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADALKKDNGEYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKANLIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTVDVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQTAEFKGTFAEA TAEAYRYADL LAKENGKYTA DLEDGGYTIN IRFAGKKVDEKPEEProtein L matrices are commercially available as Capto™ L from GEHealthcare Bio-Sciences AB, Sweden (Capto L data file 29-0100-08 AC,2014) and can be used for separation of kappa light chain-containingproteins such as intact antibodies, Fab fragments, scFv fragments,domain antibodies etc. About 75% of the antibodies produced by healthyhumans have a kappa light chain and many therapeutic monoclonalantibodies and antibody fragments contain kappa light chains.

Any bioprocess chromatography application requires comprehensiveattention to definite removal of contaminants. Such contaminants can forexample be non-eluted molecules adsorbed to the stationary phase ormatrix in a chromatographic procedure, such as non-desired biomoleculesor microorganisms, including for example proteins, carbohydrates,lipids, bacteria and viruses. The removal of such contaminants from thematrix is usually performed after a first elution of the desired productin order to regenerate the matrix before subsequent use. Such removalusually involves a procedure known as cleaning-in-place (CIP), whereinagents capable of eluting contaminants from the stationary phase areused. One such class of agents often used with chromatography media isalkaline solutions that are passed over the matrix. At present the mostextensively used cleaning and sanitizing agent is NaOH, and it isdesirable to use it in concentrations ranging from 0.05 up to e.g. 1 M,depending on the degree and nature of contamination. Protein L ishowever a rather alkali-sensitive protein compared to e.g. Protein A andonly tolerates up to about 15 mM NaOH over a large number of cycles.This means that additional, less desirable cleaning solutions, e.g. ureaor guanidinium salts, may also have to be used in order to ensuresufficient cleaning.

An extensive research has earlier been focused on the development ofengineered protein A ligands that exhibit an improved capacity towithstand alkaline pH-values. For example, WO2003/080655A1 disclosesthat Protein A domains with particular asparagine mutations areconsiderably more alkali stable than the native protein.

There is thus still a need in this field to obtain a separation matrixcontaining Protein L-derived ligands having an improved stabilitytowards alkaline cleaning procedures.

SUMMARY OF THE INVENTION

One aspect of the invention is to provide a polypeptide with improvedalkaline stability. This is achieved with a polypeptide as defined inclaim 1.

One advantage is that the alkaline stability is improved over Protein Land the parental polypeptides. A further advantage is that the highlyselective binding towards kappa light chain-containing proteinsdemonstrated for Protein L is retained in the polypeptides of theinvention.

A second aspect of the invention is to provide a nucleic acid or avector encoding a polypeptide or multimer with improved alkalinestability. This is achieved with a nucleic acid or vector as defined inthe claims.

A third aspect of the invention is to provide an expression systemcapable of expressing a polypeptide or multimer with improved alkalinestability. This is achieved with an expression system as defined in theclaims.

A fourth aspect of the invention is to provide a separation matrixcapable of selectively binding kappa light chain-containing proteins andexhibiting an improved alkaline stability. This is achieved with aseparation matrix as defined in the claims.

A fifth aspect of the invention is to provide an efficient andeconomical method of isolating a kappa light chain-containing protein.This is achieved with a method as defined in the claims.

Further suitable embodiments of the invention are described in thedependent claims.

Definitions

The terms “antibody” and “immunoglobulin” are used interchangeablyherein, and are understood to include also fragments of antibodies,fusion proteins comprising antibodies or antibody fragments andconjugates comprising antibodies or antibody fragments.

The terms a “kappa light chain-binding polypeptide” and “kappa lightchain-binding protein” herein mean a polypeptide or proteinrespectively, capable of binding to a subclass 1, 3 or 4 kappa lightchain of an antibody (also called V_(κI), V_(κIII) and V_(κIV), as in BH K Nilson et al: J. Biol. Chem. 267, 2234-2239, 1992), and include e.g.Protein L, and any variant, fragment or fusion protein thereof that hasmaintained said binding property.

The term “kappa light chain-containing protein” is used as a synonym of“immunoglobulin kappa light chain-containing protein” and herein means aprotein comprising a subclass 1, 3 or 4 kappa light chain (also calledV_(κI), V_(κIII) and V_(κIV), as in B H K Nilson et al: J. Biol. Chem.267, 2234-2239, 1992) derived from an antibody and includes any intactantibodies, antibody fragments, fusion proteins, conjugates orrecombinant proteins containing a subclass 1, 3 or 4 kappa light chain.

BRIEF DESCRIPTION OF FIGURES

FIG. 1 shows an alignment of the five kappa light chain-binding domainsof Protein L as described in U.S. Pat. No. 6,822,075 and W Kastern etal: J Biol. Chem. 267, 12820-12825, 1992.

FIG. 2 shows the alkali stability of different kappa light chain-bindingdomains of Protein L.

FIG. 3 shows the alkali stability of mutated kappa light chain-bindingdomains of Protein L.

FIG. 4 shows the alkali stability of Protein L ligands comprising fourdomains.

FIG. 5 shows the alkali stability of mutated dimeric, tetrameric andhexameric kappa light chain-binding domains of Protein L in comparisonwith Protein L.

DETAILED DESCRIPTION OF EMBODIMENTS

In one aspect the present invention discloses a kappa lightchain-binding polypeptide comprising or consisting essentially of one ormore binding domains of Peptostreptococcus magnus Protein L, whereineach of these domains is selected from the group consisting of Domain 2,Domain 3 and Domain 4. Domain 2 can have an amino acid sequence definedby SEQ ID NO:3 or SEQ ID NO:12, or it can have at least 90%, such as atleast 95%, sequence homology with SEQ ID NO:3 or 12. SEQ ID NO: 12 is avariant of SEQ ID NO: 3, with an alanine in position 31.

Domain 3 can have an amino acid sequence defined by SEQ ID NO:4, or itcan have at least 90%, such as at least 95%, sequence homology with SEQID NO:4. Domain 4 can have an amino acid sequence defined by SEQ IDNO:5, or it can have at least 90%, such as at least 95%, sequencehomology with SEQ ID NO:5.

In some embodiments of the polypeptide, each domain is selected from thegroup consisting of Domain 3 and Domain 4, or each of the domains isDomain 3. Specifically, the polypeptide may comprise or consistessentially of a multimer of Domain 3.

In certain embodiments, at least two of the domains are selected fromthe group consisting of Domain 2, Domain 3 and Domain 4, or from thegroup consisting of Domain 3 and Domain 4.

In some embodiments, the polypeptide does not contain any Domain 1 ofPeptostreptococcus Protein L. Domain 1 can have an amino acid sequenceas defined by SEQ ID NO:2, or it can have at least 90%, such as at least95% sequence homology with SEQ ID NO:2.

In certain embodiments of the polypeptide, at least the amino acid atthe position corresponding to position 45 in SEQ ID NO:2-5 (e.g. theamino acid at position 45 in SEQ ID NO: 2-5 or 12) in one or more, suchas all, of the binding domains has been mutated to an amino acid whichis not asparagine, proline or cysteine. The amino acid at position 45can e.g. be mutated to an alanine.

In some embodiments of the polypeptide, at least the amino acid at theposition corresponding to position 10 in SEQ ID NO:2-5 (e.g. the aminoacid at position 10 in SEQ ID NO: 2-5 or 12) in one or more, such asall, of the binding domains has been mutated to an amino acid which isnot asparagine, proline or cysteine. The amino acid at position 10 cane.g. be mutated to a glutamine.

In certain embodiments of the polypeptide, at least the amino acid atthe position corresponding to position 60 in SEQ ID NO:2-5 (e.g. theamino acid at position 60 in SEQ ID NO: 2-5 or 12) in one or more, suchas all, of the binding domains has been mutated to an amino acid whichis not asparagine, proline or cysteine. The amino acid at position 60can e.g. be mutated to a glutamine.

Specifically, one or more, such as all, of the binding domains may havemutations selected from the group consisting of N10Q; N45A; N60Q;N10Q,N45A; N45A,N60Q, N10Q,N60Q and N10Q,N45A,N60Q, or alternativelyselected from the group consisting of N45A; N10Q,N45A; N45A,N60Q andN10Q,N45A,N60Q.

In some embodiments of the polypeptide, at least the amino acid at theposition corresponding to position 19 in SEQ ID NO:2-5 (e.g. the aminoacid at position 19 in SEQ ID NO: 2-5 or 12) in one or more, such asall, of the binding domains has been mutated to an amino acid which isnot glutamine, asparagine, proline or cysteine. The amino acid atposition 19 can e.g. be mutated to a glutamic acid or an alanine.Specifically, one or more, such as all, of the binding domains may havemutations selected from the group consisting of Q19E and Q19A.

In certain embodiments of the polypeptide, one or more, such as all, ofsaid binding domains has an amino acid sequence selected from the groupconsisting of sequences defined by SEQ ID NO:7, SEQ ID NO:8, SEQ IDNO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:13 and SEQ ID NO:14. One ormore, such as all, of said binding domains can alternatively have anamino acid sequence selected from the group consisting of sequencesdefined by SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10 and SEQID NO:11. The polypeptide may further at the N-terminus comprise aplurality of amino acid residues originating from the cloning process orconstituting a residue from a cleaved off signaling sequence. The numberof additional amino acid residues may e.g. be 15 or less, such as 10 orless or 5 or less. As a specific example, the polypeptide may comprisean AQV sequence at the N-terminus.

(Domain 3, N45A mutation) SEQ ID NO: 7PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLN IKFAGKEKTPEE (Domain 3, N10Q, N45A mutation)SEQ ID NO: 8 PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLN IKFAGKEKTPEE (Domain 3, N45A, N60Q mutation)SEQ ID NO: 9 PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE (Domain 3, N10Q, N60Q mutation)SEQ ID NO: 10 PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKENGKYTV DVADKGYTLQ IKFAGKEKTPEE (Domain 3, N10Q, N45A, N60Q mutation)SEQ ID NO: 11  PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE (variant of Domain 2) SEQ ID NO: 12PKEEVTIKAN LIYADGKTQT AEFKGTFEEA AAEAYRYADALKKDNGEYTV DVADKGYTLN IKFAGKEKTPEE (Domain 3, Q19A mutation)SEQ ID NO: 13 PKEEVTIKAN LIYADGKTAT AEFKGTFEEA TAEAYRYADLLAKENGKYTV DVADKGYTLN IKFAGKEKTPEE (Domain 3, Q19E mutation)SEQ ID NO: 14 PKEEVTIKAN LIYADGKTET AEFKGTFEEA TAEAYRYADLLAKENGKYTV DVADKGYTLN IKFAGKEKTPEE

In some embodiments, the polypeptide is a multimer comprising, orconsisting essentially of, a plurality of mutated or non-mutated domainsas defined by any embodiment disclosed above. The multimer can e.g. be adimer, a trimer, a tetramer, a pentamer or a hexamer. It can be ahomomultimer, where all the units in the multimer are identical or itcan be a heteromultimer, where at least one unit differs from theothers. Advantageously, all the units in the multimer are alkali stable,such as by comprising the mutations disclosed above. The domains can belinked to each other directly by peptide bonds between the C- andN-termini of the domains. Alternatively, two or more units in themultimer can be linked by elements comprising oligomeric or polymericspecies, such as elements comprising up to 15 or 30 amino acids, such as1-5, 1-10 or 5-10 amino acids. The nature of such a link shouldpreferably not destabilize the spatial conformation of the domains. Thiscan e.g. be achieved by avoiding the presence of cysteine in the links.Furthermore, said link should preferably also be sufficiently stable inalkaline environments not to impair the properties of the domains. Forthis purpose, it is advantageous if the links do not contain asparagine.It can additionally be advantageous if the links do not containglutamine. The multimer may further at the N-terminus comprise aplurality of amino acid residues originating from the cloning process orconstituting a residue from a cleaved off signaling sequence. The numberof additional amino acid residues may e.g. be 15 or less, such as 10 orless or 5 or less. As a specific example, the multimer may comprise anAQV sequence at the N-terminus.

In certain embodiments, the multimer may comprise, or consistessentially, of a sequence selected from the group consisting of: SEQ IDNO: 15, SEQ ID NO: 16, SEQ ID NO: 17 and SEQ ID NO: 18, such as asequence selected from the group consisting of SEQ ID NO: 16, SEQ ID NO:17 and SEQ ID NO: 18.

(Domain 3, tetramer) SEQ ID NO: 15PKEEVTIKAN LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKENGKYTV DVADKGYTLN IKFAGKEKTPEE PKEEVTIKANLIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKENGKYTVDVADKGYTLN IKFAGKEKTPEE PKEEVTIKAN LIYADGKTQTAEFKGTFEEA TAEAYRYADL LAKENGKYTV DVADKGYTLNIKFAGKEKTPEE PKEEVTIKAN LIYADGKTQT AEFKGTFEEATAEAYRYADL LAKENGKYTV DVADKGYTLN IKFAGKEKTPEEDomain 3(N10Q, N45A, N60Q)2 SEQ ID NO: 16PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQLIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEEDomain 3 (N10Q, N45A, N60Q)4 SEQ ID NO: 17PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQLIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTVDVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQTAEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQIKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEATAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEEDomain 3 (N10Q, N45A, N60Q)6 SEQ ID NO: 18PKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQLIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTVDVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQTAEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQIKFAGKEKTPEE PKEEVTIKAQ LIYADGKTQT AEFKGTFEEATAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEEPKEEVTIKAQ LIYADGKTQT AEFKGTFEEA TAEAYRYADLLAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE PKEEVTIKAQLIYADGKTQT AEFKGTFEEA TAEAYRYADL LAKEAGKYTV DVADKGYTLQ IKFAGKEKTPEE

In some embodiments, the polypeptide and/or multimer, as disclosedabove, further comprises at the C-terminus or N-terminus one or morecoupling elements, selected from the group consisting of a cysteineresidue, a plurality of lysine residues and a plurality of histidineresidues. The coupling element may e.g. be a single cysteine at theC-terminus. The coupling element(s) may be directly linked to the C- orN-terminus, or it/they may be linked via a linker comprising up to 15amino acids, such as 1-5, 1-10 or 5-10 amino acids. This stretch shouldpreferably also be sufficiently stable in alkaline environments not toimpair the properties of the mutated protein. For this purpose, it isadvantageous if the stretch does not contain asparagine. It canadditionally be advantageous if the stretch does not contain glutamine.An advantage of having a C- or N-terminal cysteine is that endpointcoupling of the protein can be achieved through reaction of the cysteinethiol with an electrophilic group on a support. This provides excellentmobility of the coupled protein which is important for the bindingcapacity.

The alkali stability of the polypeptide or multimer can be assessed bycoupling it to an SPR chip, e.g. to Biacore CM5 sensor chips asdescribed in the examples, and measuring the kappa light chain-bindingcapacity of the chip, using e.g. a specific kappa light chain-containingprotein or polyclonal human IgG (where the majority of the IgG moleculeshave a kappa light chain), before and after incubation in alkalinesolutions at a specified temperature, e.g. 22+/−2° C. The incubation cane.g. be performed in 0.1 M NaOH for a number of 10 min cycles, such as50, 96 or 100 cycles. The binding capacity of the matrix after 96-100 10min incubation cycles in 0.1 M NaOH at 22+/−2° C. can be at least 40,such as at least 50, or at least 55% of the binding capacity before theincubation. Alternatively, the remaining binding capacity after 96-100cycles for a particular mutant measured as above can be compared withthe remaining binding capacity for the parental polypeptide/multimer. Inthis case, the remaining binding capacity for the mutant may be at least105%, such as at least 110%, at least 125%, at least 150% or at least200% of the parental polypeptide/multimer.

The invention also discloses a kappa light chain-binding polypeptidecomprising at least one mutated binding domain of PeptostreptococcusProtein L, in which at least one asparagine residue of a parental domaindefined by, or having at least 95% or 98% sequence homology with, SEQ IDNO: 2-6 or 12 has been mutated to another amino acid residue which isnot asparagine, proline or cysteine. The polypeptide may comprise atleast the mutation N45A and/or the mutation N60Q. In specificembodiments, the mutation(s) are selected from the group consisting ofN45A; N10Q,N45A; N45A,N60Q, N10Q,N60Q and N10Q,N45A,N60Q, oralternatively selected from the group consisting of N45A; N10Q,N45A;N45A,N60Q and N10Q,N45A,N60Q. The alkali stability relative to aparental polypeptide can be improved and measured as disclosed above.

In some embodiments the polypeptide comprises or consists essentially ofa plurality of mutated binding domains, such as 2, 3, 4, 5 or 6 domains,wherein each domain comprises at least one of the mutations N10Q, N45Aand N60Q, such as N45A and/or N60Q. Specifically, the mutation(s) ineach domain can be selected from the group consisting of N45A;N10Q,N45A; N45A,N60Q, N10Q,N60Q and N10Q,N45A,N60Q, or alternativelyselected from the group consisting of N45A; N10Q,N45A; N45A,N60Q andN10Q,N45A,N60Q. The domains can optionally be linked to each other byelements comprising up to 15 amino acids.

In a second aspect the present invention discloses a nucleic acidencoding a polypeptide or multimer according to any embodiment disclosedabove. Thus, the invention encompasses all forms of the present nucleicacid sequence such as the RNA and the DNA encoding the polypeptide ormultimer. The invention embraces a vector, such as a plasmid, which inaddition to the coding sequence comprises the required signal sequencesfor expression of the polypeptide or multimer according the invention.In one embodiment, the vector comprises nucleic acid encoding a multimeraccording to the invention, wherein the separate nucleic acids encodingeach unit may have homologous or heterologous DNA sequences.

In a third aspect the present invention discloses an expression system,which comprises, a nucleic acid or a vector as disclosed above. Theexpression system may e.g. be a gram-positive or gram-negativeprokaryotic host cell system, e.g. Bacillus sp. or Escherichia coliwhich has been modified to express the present polypeptide or multimer.In an alternative embodiment, the expression system is a eukaryotic hostcell system, such as a yeast, e.g. Pichea pastoris or Saccharomycescerevisiae.

In a fourth aspect, the present invention discloses a separation matrix,wherein a plurality of polypeptides or multimers according to anyembodiment disclosed above have been coupled to a solid support. Such amatrix is useful for separation of kappa light chain-containing proteinsand, due to the improved alkali stability of the polypeptides/multimers,the matrix will withstand highly alkaline conditions during cleaning,which is essential for long-term repeated use in a bioprocess separationsetting. The alkali stability of the matrix can be assessed by measuringthe kappa light chain-binding capacity, using e.g. a specific kappalight chain-containing protein or polyclonal human IgG, before and afterincubation in alkaline solutions at a specified temperature, e.g.22+/−2° C. The incubation can e.g. be performed in 0.1 M NaOH for anumber of 15 min cycles, such as 100, 200 or 300 cycles. The bindingcapacity of the matrix after 100 15 min incubation cycles in 0.1 M NaOHat 22+/−2° C. can be at least 80, such as at least 85, at least 90 or atleast 95% of the binding capacity before the incubation. Alternatively,the incubation can be performed in 0.1 M NaOH for a number of 4 hcycles, such as 6 cycles giving a total incubation time of 24 h. Thebinding capacity of the matrix after 24 h min total incubation time in0.1 M NaOH at 22+/−2° C. can be at least 80, such as at least 85, atleast 90 or at least 95% of the binding capacity before the incubation.

As the skilled person will understand, the expressed polypeptide ormultimer should be purified to an appropriate extent before beenimmobilized to a support. Such purification methods are well known inthe field, and the immobilization of protein-based ligands to supportsis easily carried out using standard methods. Suitable methods andsupports will be discussed below in more detail.

The solid support of the matrix according to the invention can be of anysuitable well-known kind. A conventional affinity separation matrix isoften of organic nature and based on polymers that expose a hydrophilicsurface to the aqueous media used, i.e. expose hydroxy (—OH), carboxy(—COOH), carboxamido (—CONH₂, possibly in N-substituted forms), amino(—NH₂, possibly in substituted form), oligo- or polyethylenoxy groups ontheir external and, if present, also on internal surfaces. The solidsupport can suitably be porous. The porosity can be expressed as a Kayor Kd value (the fraction of the pore volume available to a probemolecule of a particular size) measured by inverse size exclusionchromatography, e.g. according to the methods described in GelFiltration Principles and Methods, Pharmacia LKB Biotechnology 1991, pp6-13. By definition, both Kd and Kay values always lie within the range0-1. The Kay value can advantageously be 0.6-0.95, e.g. 0.7-0.90 or0.6-0.8, as measured with dextran of Mw 110 kDa as a probe molecule. Anadvantage of this is that the support has a large fraction of pores ableto accommodate both the polypeptides/multimers of the invention andimmunoglobulins binding to the polypeptides/multimers and to providemass transport of the immunoglobulins to and from the binding sites.

The polypeptides or multimers may be attached to the support viaconventional coupling techniques utilising e.g. thiol, amino and/orcarboxy groups present in the ligand. Bisepoxides, epichlorohydrin,CNBr, N-hydroxysuccinimide (NHS) etc. are well-known coupling reagents.Between the support and the polypeptide/multimer, a molecule known as aspacer can be introduced, which improves the availability of thepolypeptide/multimer and facilitates the chemical coupling of thepolypeptide/multimer to the support. Suitable spacers can be introducede.g. by activation of the support with epichlorohydrin, butanedioldiepoxide, allyl glycidyl ether etc. Alternatively, thepolypeptide/multimer may be attached to the support by non-covalentbonding, such as physical adsorption or biospecific adsorption.

In some embodiments the matrix comprises 5-20, such as 5-15 mg/ml, 5-11mg/ml or 8-11 mg/ml of the polypeptide or multimer coupled to thesupport. The amount of coupled polypeptide/multimer can be controlled bythe concentration of polypeptide/multimer used in the coupling process,by the coupling conditions used and/or by the pore structure of thesupport used. As a general rule the absolute binding capacity of thematrix increases with the amount of coupled polypeptide/multimer, atleast up to a point where the pores become significantly constricted bythe coupled polypeptide/multimer. The relative binding capacity per mgcoupled polypeptide/multimer will decrease at high coupling levels,resulting in a cost-benefit optimum within the ranges specified above.

In some embodiments the polypeptides are coupled to the support viamultipoint attachment. This can suitably be done by using such couplingconditions that a plurality of reactive groups in the polypeptide reactwith reactive groups in the support. Typically, multipoint attachmentcan involve the reaction of several intrinsic reactive groups of aminoacid residues in the sequence, such as amines in lysines, with thereactive groups on the support, such as epoxides, cyanate esters (e.g.from CNBr activation), succinimidyl esters (e.g. from NHS activation)etc. It is however also possible to deliberately introduce reactivegroups at different positions in the polypeptides to affect the couplingcharacteristics. In order to provide multipoint coupling via lysines,the coupling reaction is suitably carried out at a pH where asignificant fraction of the lysine primary amines are in thenon-protonated nucleophilic state, e.g. at pH higher than 8.0, such asabove 10.

In certain embodiments the polypeptides or multimers are coupled to thesupport via thioether bonds. Methods for performing such coupling arewell-known in this field and easily performed by the skilled person inthis field using standard techniques and equipment. Thioether bonds areflexible and stable and generally suited for use in affinitychromatography. In particular when the thioether bond is via a terminalor near-terminal cysteine residue on the polypeptide or multimer, themobility of the coupled polypeptide/multimer is enhanced which providesimproved binding capacity and binding kinetics. In some embodiments thepolypeptide/multimer is coupled via a C-terminal cysteine provided onthe protein as described above. This allows for efficient coupling ofthe cysteine thiol to electrophilic groups, e.g. epoxide groups,halohydrin groups etc. on a support, resulting in a thioether bridgecoupling. The polypeptide/multimer can e.g. be coupled via single-pointattachment, e.g. via a single cysteine or by directed multipointattachment, using e.g. a plurality of lysines or other coupling groupsnear a terminus of the polypeptide/multimer.

In certain embodiments the support comprises a polyhydroxy polymer, suchas a polysaccharide. Examples of polysaccharides include e.g. dextran,starch, cellulose, pullulan, agar, agarose etc. Polysaccharides areinherently hydrophilic with low degrees of nonspecific interactions,they provide a high content of reactive (activatable) hydroxyl groupsand they are generally stable towards alkaline cleaning solutions usedin bioprocessing.

In some embodiments the support comprises agar or agarose. The supportsused in the present invention can easily be prepared according tostandard methods, such as inverse suspension gelation (S Hjertén:Biochim Biophys Acta 79(2), 393-398 (1964). Alternatively, the basematrices are commercially available products, such as crosslinkedagarose beads sold under the name of SEPHAROSE™ FF (GE Healthcare). Inan embodiment, which is especially advantageous for large-scaleseparations, the support has been adapted to increase its rigidity usingthe methods described in U.S. Pat. No. 6,602,990 or U.S. Pat. No.7,396,467, which are hereby incorporated by reference in their entirety,and hence renders the matrix more suitable for high flow rates.

In certain embodiments the support, such as a polysaccharide or agarosesupport, is crosslinked, such as with hydroxyalkyl ether crosslinks.Crosslinker reagents producing such crosslinks can be e.g.epihalohydrins like epichlorohydrin, diepoxides like butanedioldiglycidyl ether, allylating reagents like allyl halides or allylglycidyl ether. Crosslinking is beneficial for the rigidity of thesupport and improves the chemical stability. Hydroxyalkyl ethercrosslinks are alkali stable and do not cause significant nonspecificadsorption.

Alternatively, the solid support is based on synthetic polymers, such aspolyvinyl alcohol, polyhydroxyalkyl acrylates, polyhydroxyalkylmethacrylates, polyacrylamides, polymethacrylamides etc. In case ofhydrophobic polymers, such as matrices based on divinyl andmonovinyl-substituted benzenes, the surface of the matrix is oftenhydrophilised to expose hydrophilic groups as defined above to asurrounding aqueous liquid. Such polymers are easily produced accordingto standard methods, see e.g. “Styrene based polymer supports developedby suspension polymerization” (R Arshady: Chimica e L′Industria 70(9),70-75 (1988)). Alternatively, a commercially available product, such asSOURCE™ (GE Healthcare) is used. In another alternative, the solidsupport according to the invention comprises a support of inorganicnature, e.g. silica, zirconium oxide etc.

In yet another embodiment, the solid support is in another form such asa surface, a chip, capillaries, or a filter (e.g. a membrane or a depthfilter matrix).

As regards the shape of the matrix according to the invention, in oneembodiment the matrix is in the form of a porous monolith. In analternative embodiment, the matrix is in beaded or particle form thatcan be porous or non-porous. Matrices in beaded or particle form can beused as a packed bed or in a suspended form. Suspended forms includethose known as expanded beds and pure suspensions, in which theparticles or beads are free to move. In case of monoliths, packed bedand expanded beds, the separation procedure commonly followsconventional chromatography with a concentration gradient. In case ofpure suspension, batch-wise mode will be used.

In a sixth aspect, the present invention discloses a method of isolatinga kappa light chain-containing protein, wherein a separation matrix asdisclosed above is used.

In certain embodiments, the method comprises the steps of:

a) contacting a liquid sample comprising a kappa light chain-containingprotein with a separation matrix as disclosed above,

b) washing said separation matrix with a washing liquid,

c) eluting the kappa light chain-containing protein from the separationmatrix with an elution liquid, and

d) cleaning the separation matrix with a cleaning liquid.

The method may also comprise steps of, before step a), providing anaffinity separation matrix according to any of the embodiments describedabove and providing a solution comprising a kappa light chain-containingprotein and at least one other substance as a liquid sample and of,after step c), recovering the eluate and optionally subjecting theeluate to further separation steps, e.g. by anion or cation exchangechromatography, multimodal chromatography and/or hydrophobic interactionchromatography. Suitable compositions of the liquid sample, the washingliquid and the elution liquid, as well as the general conditions forperforming the separation are well known in the art of affinitychromatography and in particular in the art of Protein L chromatography.The liquid sample comprising a kappa light chain-containing protein andat least one other substance may comprise host cell proteins (HCP), suchas chinese hamster ovary (CHO) cell, E. coli or yeast cell proteins.Contents of CHO cell and E. coli proteins can conveniently be determinedby immunoassays directed towards these proteins, e.g. the CHO HCP or E.coli HCP ELISA kits from Cygnus Technologies. The host cell proteins orCHO cell/E. coli/yeast proteins may be desorbed during step b).

The elution may be performed by using any suitable solution used forelution from Protein L media. This can e.g. be a solution or buffer withpH 4 or lower, such as pH 2.5-4 or 2.8-3.5. In some embodiments theelution buffer or the elution buffer gradient comprises at least onemono- di- or trifunctional carboxylic acid or salt of such a carboxylicacid. In certain embodiments the elution buffer or the elution buffergradient comprises at least one anion species selected from the groupconsisting of acetate, citrate, glycine, succinate, phosphate, andformiate.

In some embodiments, the cleaning liquid is alkaline, such as with a pHof 12-14. Such solutions provide efficient cleaning of the matrix, inparticular at the upper end of the interval

In certain embodiments, the cleaning liquid comprises 0.01-1.0 M NaOH orKOH, such as 0.05-1.0 or 0.05-0.1 M NaOH or KOH. The high stability ofthe polypeptides of the invention enables the use of such comparativelystrong alkaline solutions.

In some embodiments, steps a)-d) are repeated at least 10 times, such asat least 50 times or 50-200 times. This is important for the processeconomy in that the matrix can be re-used many times.

EXAMPLES

Mutagenesis of Protein

Monomer constructs were designed from a Protein L disclosed in U.S. Pat.No. 6,822,075 (SEQ ID NO: 1), containing four kappa light chain-bindingdomains. These are numbered 1, 2, 3 and 4, starting from the N-terminus(FIG. 1). The DNA fragments were purchased from a DNA synthesizingcompany (DNA2.0). Four monomer constructs were prepared in apJexpress201 cloning vector, each with an N-terminal cysteine. For anoverview of constructs, see SEQ ID NO: 2,4,5,12. Constructs weresubcloned to expression vector pGO, containing E. coli GAP promoter andOmpA signal peptide sequence for periplasmatic localization of thetarget protein. The sequence encoding the four domains was prepared byamplifying with oligonucleotides containing the restriction enzymerecognition sites for FspI and PstI on the 5’ side and 3′ side,respectively. The prepared DNA fragment encoding each domain wasdigested with FspI and PstI (New England Biolabs). Separately,expression vector was prepared with digestion with FspI and PstI andpurified by agarose gel electrophoresis and recovered. Both were mixedand ligated with Quick ligation kit (New England Biolabs). The ligatedplasmid expressing each domain was transformed into a chemical competentE. coli K12 strain with a heat shock method.

Further mutations of amino acids N10, N45, Q19, and N60 in domain 3 wereprepared in expression vector pJexpress401(DNA2.0) containing T5promoter under a lac operon control mechanism (SEQ ID NO: 7-11,13-14).Constructs were designed with and without OmpA signal peptide butwithout a C-terminal cysteine.

Tetramers of domain 3, dimer, tetramer and hexamer of domain 3 with N45,N10 and N60 mutations were also prepared in pJexpress401, with andwithout C-terminal cysteine (SEQ ID NO: 15-18).

Construct Expression and Purification

The E. coli K12 recombinant cells were cultured in shake flasks withLB-broth (10 g peptone, 5 g yeast extract, 5 g NaCl) supplemented with25 mg/l kanamycin at 37° C. until optical density at 600 nm reached 0.8.At this point protein expression was induced with Isopropylβ-D-1-thiogalactopyranoside (VWR International) with final concentrationof 1 mM. Upon induction the temperature was lowered to 30° C. and thecultures were incubated for 5 hours. The cultivation was stopped andcells were centrifuged for 15 minutes at 4000×g and the supernatant wasdiscarded. Cells were resuspended in 1/10 of culturing volume withphosphate buffered saline (PBS) and sonicated using pulse-sonicationwith an active time of 2 minutes. The sonicated samples were clarifiedfrom cell debris by centrifugation at 6000×g for 30 minutes, followed bymicrofiltration with a membrane having a 0.2 μm pore size.

The purified ligands were analyzed with LC-MS to determine the purityand to ascertain that the molecular weight corresponded to the expected(based on the amino acid sequence).

Example 1

The purified monomeric ligands listed in Table 1, further comprising, inthe cases of the non-mutated single domains, a cysteine at the Cterminus and an AQV sequence at the N-terminus, were immobilized onBiacore CMS sensor chips (GE Healthcare, Sweden), using the aminecoupling kit of GE Healthcare (for carbodiimide coupling of amines onthe carboxymethyl groups on the chip) in an amount sufficient to give asignal strength of about 1000RU in a Biacore instrument (GE Healthcare,Sweden). To follow the IgG binding capacity of the immobilized surface 1mg/ml human polyclonal IgG (Gammanorm) was flowed over the chip and thesignal strength was noted. The surface was then cleaned-in-place (CIP),i.e. flushed with 100 mM NaOH for 10 minutes at room temperature(22+/−2° C.). This was repeated for 96 cycles and the immobilized ligandalkaline stability was followed as the relative loss of IgG bindingcapacity (signal strength) after each cycle. The results for thenon-mutated domains are shown in FIG. 2 and indicate that Domain 1 has adistinctly lower alkali stability than the other domains and that Domain3 has the highest alkali stability. Results for single-domain asparaginemutants of Domain 3 are shown in FIG. 3 and show an improved alkalistability for all the mutants in comparison with the parental Domain 3,which was used as a reference in parallel with the mutations.

TABLE 1 Retained capacity Ref. Sample/ after 96 capacity ref. LigandSequence cycles (%) (%) ratio Domain 1 (D1) SEQ ID NO: 2 13 31 0.42Domain 2 (D2) SEQ ID NO: 12 22 31 0.71 Domain 3 (D3) SEQ ID NO: 4 31 311.00 Domain 4 (D4) SEQ ID NO: 5 26 31 0.84 D3(N45A)1 SEQ ID NO: 7 44 311.42 D3(N10Q, N45A)1 SEQ ID NO: 8 48 31 1.55 D3(N45A, N60Q)1 SEQ ID NO:9 59 31 1.90 D3(N10Q, SEQ ID NO: 11 59 31 1.90 N45A, N60Q)1 Domain 3(D3) SEQ ID NO: 4 28 28 1.00 D3(Q19A)1 SEQ ID NO: 13 28 28 1.00D3(Q19E)1 SEQ ID NO: 14 31 28 1.11

Example 2

The purified multidomain ligands listed in Table 2 were immobilized onBiacore CMS sensor chips (GE Healthcare, Sweden), using the aminecoupling kit of GE Healthcare (for carbodiimide coupling of amines onthe carboxymethyl groups on the chip) in an amount sufficient to give asignal strength of about 1000RU in a Biacore instrument (GE Healthcare,Sweden). The Protein L had an additional AIHNRA sequence at theN-terminus. To follow the IgG binding capacity of the immobilizedsurface 1 mg/ml human polyclonal IgG (Gammanorm) was flowed over thechip and the signal strength was noted. The surface was thencleaned-in-place (CIP), i.e. flushed with 100 mM NaOH for 10 minutes atroom temperature (22+/−2° C.). This was repeated for 96 cycles and theimmobilized ligand alkaline stability was followed as the relative lossof IgG binding capacity (signal strength) after each cycle. The resultsare shown in Table 2 and FIG. 4 and show that the tetrameric Domain 3has an improved alkali stability in comparison with Protein L which wasrun in parallel as a reference.

TABLE 2 Retained capacity Ref. after 96 capacity Sample/ref. LigandSequence cycles (%) (%) ratio Protein L SEQ ID NO: 1 28 28 1.00 Domain 3SEQ ID NO: 15 38 28 1.36 tetramer

Example 3

The purified multidomain ligands listed in Table 3 were immobilized onBiacore CM5 sensor chips and evaluated by the methods used in Example 2.—cys at the end of the ligand designation indicates that the ligand hasa C-terminal cysteine in addition to the sequence defined by SEQ ID NO:16-18. The results are shown in Table 3 and FIG. 5 and show that all themutated Domain 3 dimers, tetramers and hexamers have an improved alkalistability in comparison with Protein L which was run in parallel as areference.

TABLE 3 Retained capacity Ref. after 100 capacity Sample/ref. LigandSequence cycles (%) (%) ratio Protein L SEQ ID NO: 1 23 23 1.00 D3(N10Q,N45A, SEQ ID NO: 16 59 23 2.56 N60Q)2 D3(N10Q, N45A, SEQ ID NO: 16 59 232.56 N60Q)2-cys D3(N10Q, N45A, SEQ ID NO: 17 60 23 2.61 N60Q)4 D3(N10Q,N45A, SEQ ID NO: 17 54 23 2.35 N60Q)4-cys D3(N10Q, N45A, SEQ ID NO: 1858 23 2.52 N60Q)6 D3(N10Q, N45A, SEQ ID NO: 18 56 23 2.43 N60Q)6-cys

Example 4

The purified di-, tetra- and hexameric ligands of Table 4 wereimmobilized on agarose beads using the methods described below andassessed for capacity. The results are shown in Table 4.

TABLE 4 Ligand QB10 Fab QB10 Dab Ligand Sequence content mg/ml mg/mlD3(N10Q, SEQ ID NO: 16 9.5 mg/ml 19.3 15.4 N45A, N60Q)2 D3(N10Q, SEQ IDNO: 17 8.8 mg/ml 19.3 15.9 N45A, N60Q)4 D3(N10Q, SEQ ID NO: 18 11.0mg/ml  21.1 16.7 N45A, N60Q)6

Activation

The base matrix used was rigid cross-linked agarose beads of 85micrometers (volume-weighted) median diameter, prepared according to themethods of U.S. Pat. No. 6,602,990 and with a pore size corresponding toan inverse gel filtration chromatography Kay value of 0.70 for dextranof Mw 110 kDa, according to the methods described in Gel FiltrationPrinciples and Methods, Pharmacia LKB Biotechnology 1991, pp 6-13.

25 mL (g) of drained base matrix, 10.0 mL distilled water and 2.02 gNaOH (s) was mixed in a 100 mL flask with mechanical stirring for 10 minat 25° C. 4.0 mL of epichlorohydrin was added and the reactionprogressed for 2 hours. The activated gel was washed with 10 gelsediment volumes (GV) of water.

Coupling

The activated gel was washed with 5 GV 0.2 M phosphate/1 mM EDTA pH 11.5(coupling buffer). 15 ml gel+13 mg ligand/ml gel (5.0 ml)+5.5 mlcoupling buffer+4.7 g sodium sulfate were mixed in a 50 ml flask andstirred at 30° C. for 18.5 h. The pH was measured as 10.8.

After immobilization the gels were washed 3×GV with distilled water andthen 5×GV with 0.1 M phosphate/1 mM EDTA pH 8.5. The gels +1 GV {0.1 Mphosphate/1 mM EDTA/7.5% thioglycerol pH 8.5} was mixed and the flaskwas stirred at 45° C. for 2 h 20 min. The gel was then washedalternately with 1×GV 0.1 M HAc and 1×GV {0.1 M TRIS/0.15 M NaCl pH 8.5}and and then 6×GV with distilled water. Gel samples were sent to anexternal laboratory for amino acid analysis and the ligand content(mg/ml gel) was calculated from the total amino acid content. Thecoupling protocol used provides multipoint coupling, with severallysines of each domain attached to the gel.

2 ml of resin was packed in TRICORN™ 5 100 columns.

Protein

a) Purified Fab prepared from a papain-digested IgG mAb, diluted to 1mg/ml in Equilibration buffer.

b) Purified Dab prepared from heat-treated E. coli supernatant, dilutedto 1 mg/ml in Equilibration buffer. The Dab contained solely a kappalight chain, without any antigen-binding site.

Equilibration Buffer

APB Phosphate buffer 20 mM+0.15 M NaCl, pH 7.4 (Medicago)

Adsorption Buffer

APB Phosphate buffer 20 mM +0.15 M NaCl, pH 7.4 (Medicago).

Elution Buffer

25 mM Citrate pH 2.5

The breakthrough capacity was determined with an ÄKTAExplorer 10 systemat a residence time of 4 minutes. Equilibration buffer was run throughthe bypass column until a stable baseline was obtained. This was doneprior to auto zeroing. Sample was applied to the column until a 100% UVsignal was obtained. Then, equilibration buffer was applied again untila stable baseline was obtained.

Sample was loaded onto the column until a UV signal of 85% of maximumabsorbance was reached. The column was then washed with equilibrationbuffer and eluted at pH 2.5 at a flow rate of 0.5 ml/min.

For calculation of breakthrough capacity at 10%, the equation below wasused. That is the amount of Fab/Dab that is loaded onto the column untilthe concentration of Fab/Dab in the column effluent is 10% of theFab/Dab concentration in the feed.

$q_{10\%} = {\frac{C_{0}}{V_{C}}\left\lbrack {V_{app} - V_{sys} - {\int_{V_{sys}}^{V_{app}}{\frac{{A(V)}*A_{sub}}{A_{100\%} - A_{sub}}*{dv}}}} \right\rbrack}$

-   -   A_(100%)=100% UV signal;    -   A_(sub)=absorbance contribution from non-binding proteins;    -   A(V)=absorbance at a given applied volume;    -   V_(c)=column volume;    -   V_(app)=volume applied until 10% breakthrough;    -   V_(sys)=system dead volume;    -   C₀=feed concentration.

The dynamic binding capacity (DBC) at 10% breakthrough was calculatedand the appearance of the curve was studied. The curve was also studiedregarding binding, elution and CIP peak. The dynamic binding capacity(DBC) was calculated for 10 and 80% breakthrough.

This written description uses examples to disclose the invention,including the best mode, and also to enable any person skilled in theart to practice the invention, including making and using any devices orsystems and performing any incorporated methods. The patentable scope ofthe invention is defined by the claims, and may include other examplesthat occur to those skilled in the art. Such other examples are intendedto be within the scope of the claims if they have structural elementsthat do not differ from the literal language of the claims, or if theyinclude equivalent structural elements with insubstantial differencesfrom the literal languages of the claims. All patents and patentapplications mentioned in the text are hereby incorporated by referencein their entireties, as if they were individually incorporated.

1. A kappa light chain-binding polypeptide comprising at least onemutated binding domain of Peptostreptococcus Protein L, in which domainat least one asparagine residue of a parental domain defined by, orhaving at least 95% or 98% sequence homology with, SEQ ID NOS: 2-6 or 12has been mutated to another amino acid residue which is not asparagine,proline or cysteine.
 2. The polypeptide of claim 1, comprising at leastthe mutation N45A.
 3. The polypeptide of claim 1, comprising at leastthe mutation N60Q.
 4. The polypeptide of claim 1, wherein themutation(s) are selected from the group consisting of N10Q; N45A; N60Q;N10Q,N45A; N45A,N60Q, N10Q,N60Q and N10Q,N45A,N60Q or from the groupconsisting of N45A; N10Q,N45A; N45A,N60Q, N10Q,N60Q and N10Q,N45A,N60Q.5. The polypeptide of claim 1, comprising or consisting essentially of aplurality of mutated binding domains, such as 2, 3, 4, 5 or 6 domains,wherein each domain comprises the mutation N45A and/or N60Q.
 6. Thepolypeptide of claim 5, wherein the mutation(s) in each domain areselected from the group consisting of N10Q; N45A; N60Q; N10Q,N45A;N45A,N60Q, N10Q,N60Q and N10Q,N45A,N60Q or from the group consisting ofN45A; N10Q,N45A; N45A,N60Q, N10Q,N60Q and N10Q,N45A,N60Q.
 7. Thepolypeptide of claim 5, wherein the domains are linked by elementscomprising up to 15 amino acids.
 8. The polypeptide according to claim1, wherein the alkaline stability is improved relative to a parentalpolypeptide, as measured by the remaining binding capacity for kappalight chain-containing proteins after 96-100 10 min incubation cycles in0.1 M aqueous NaOH at 22+/−2° C.
 9. The polypeptide of claim 1, whereinthe parental domain is defined by, or has at least 95% or 98% sequencehomology with, an amino acid sequence selected from the group consistingof SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO:
 12. 10. Thepolypeptide of claim 1, wherein the parental domain is defined by, orhas at least 95% or 98% sequence homology with SEQ ID NO:
 4. 11. Anucleic acid or a vector encoding a polypeptide or multimer according toclaim
 1. 12. An expression system, which comprises a nucleic acid orvector according to claim
 11. 13. A separation matrix, wherein aplurality of polypeptides of claim 1 have been coupled to a solidsupport.
 14. The separation matrix according to claim 13, wherein thepolypeptides have been coupled to the solid support by multipointattachment.
 15. The separation matrix of claim 13, wherein the bindingcapacity of the matrix for kappa light chain-containing proteins after100 10 min incubation cycles in 0.1 M NaOH at 22+/−2° C. is at least 40,such as at least 50, or at least 55% of the binding capacity before theincubation.
 16. A method of isolating a kappa light chain-containingprotein, wherein a separation matrix of claim 13 is used.
 17. A methodof isolating a kappa light chain-containing protein comprising the stepsof: a) contacting a liquid sample comprising a kappa lightchain-containing protein with a separation matrix of claim 13, b)washing said separation matrix with a washing liquid, c) eluting thekappa light chain-containing protein from the separation matrix with anelution liquid, and d) cleaning the separation matrix with a cleaningliquid.
 18. The method of claim 17, wherein the cleaning liquid isalkaline, such as with a pH of 12-14.
 19. The method of claim 17,wherein the cleaning liquid comprises 0.01-1.0 M NaOH or KOH, such as0.05-1.0 M or 0.05-0.1 M NaOH or KOH.
 20. The method of claim 17,wherein steps a)-d) are repeated at least 10 times, such as at least 50times or 50-200 times.